Sleep is a fundamental biological process conserved across the animal kingdom. Zebrafish, a small diurnal teleost extensively used in developmental biology, will be used as a model to study sleep and sleep regulatory networks. Previous studies have shown that a genuine sleep-like state exists in this species, as defined using behavioral criteria (circadian rhythm, reversible periods of immobility, place preference, characteristic posture, increased arousal threshold, sleep rebound). Moreover, sleep-wake molecular actors identified in mammals are also conserved in this species as well as hypnotic drug targets. Finally, the hypocretin (hcrt, aka orexin) system, a system involved in the pathophysiology of the sleep disorder narcolepsy, also exists in zebrafish. HCRTs are neuropeptides involved in the regulation of sleep and energy balance in mammals. We found that there are less than 50 hypocretin neurons in an adult zebrafish hypothalamus and cloned a very compact zebrafish hcrt promoter (1kb) capable of accurately mimicking the native hypocretin pattern. Moreover, we also identify a null mutation (hcrtr168) in the sole hypocretin receptor (hcrtr) present in zebrafish. Fish lacking this receptor have fragmented sleep and a 30% sleep reduction in the dark. In this proposal, we propose to identify, with a yeast one-hybrid assay, transcription factor(s) able to bind a core 13 base pair promoter element essential and sufficient for hypocretin expression (first specific aim). Further, we plan to study the zebrafish hcrt neurocircuitry to understand its sleep-promoting function (second specific aim). To do so, we will, (i) study its connections with hcrt:GFP and hcrtr:mCherry transgenic lines, (ii) study when it is active with a calcium imaging assay using hcrt:GCaMP2 and hcrtr:GCaMP2 transgenic lines, and (iii) evaluate whether hypocretin signaling is excitatory or inhibitory by analyzing neurotransmitter phenotype co-expression and the activity of the hcrtr positive neurons (hcrtr:GCaMP2) when hcrt neurons are silenced (hcrt:Kir2.1) or the hcrtr is missing (hcrtr168). Finally in the third specific aim, we will identify novel sleep- and wake-active nuclei in this species using immediate early gene (c- fos) expression analysis of adult zebrafish brains during the night and the day, after sleep deprivation and after hypnotic drug treatments. PUBLIC HEALTH RELEVANCE 15% of the population suffers of sleep disorders. However, sleep is still a poorly understood phenomenon. Our laboratory uses a simple model animal, the zebrafish, to understand the development, organization and function of a neuronal hypocretin system, responsible when disrupted of the sleep disorder Narcolepsy. As a developmental model, zebrafish will help us to understand how hypocretin expression can be lost in humans and, and as a neurobiology/sleep model, it will help us to decipher the underlying molecular and cellular mechanisms of sleep, and to generate the basic knowledge indispensable for future efficient therapies.